Summary: The overall goal of this proposal is to understand how mammalian cells detect and respond to the presence of intracellular bacterial pathogens. Despite antibiotics, bacterial infections continue to present a significant public health challenge. My studies focus on the gram-negative intracellular pathogen Legionella pneumophila, the causative agent of a severe pneumonia called Legionnaires' Disease. The virulence of Legionella depends on its unique ability to survive and grow within macrophages. Previous work has established that a gene called Naip5 is instrumental in orchestrating cellular defenses that protect macrophages from Legionella infection, but the molecular mechanism by which Naip5 confers resistance to Legionella has remained largely mysterious. Naip5 exhibits homology to a large family of cytosolic pathogen-detector proteins that also includes Ipaf, Nods and Nalps. Our preliminary results suggest that resistance to Legionella depends on activation of a rapid caspase-1-mediated cell death that is triggered upon the detection of bacterial flagellin in the macrophage cytosol. Thus, the specific aims of this grant proposal are: 1. To test the hypothesis that Naip5 restricts Legionella growth in by initiating a rapid caspase-1 dependent macrophage cell death. 2. Test the hypothesis that there is a cytosolic surveillance system that detects flagellin and induces rapid macrophage cell death. 3. Generate and characterize the phenotype of a Naip5 knockout mouse using in vitro and in vivo models 4. Develop a brightly luminescent strain of Legionella to permit high throughput screening assays to identify host proteins required for restricting (or permitting) Legionella growth. Relevance: It is anticipated that results obtained from the above work will permit a deeper understanding of how bacteria cause disease and of what factors lead to successful immune responses to these bacteria. Such knowledge should contribute to rational approaches to designing novel antibacterial therapies. [unreadable] [unreadable] [unreadable]